Protein drug conjugates such as antibody drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. ADCs, for example, consist of a tumor antigen-specific antibody that is coupled to a chemotherapeutic small molecule cytotoxin. Through targeted delivery of potent cytotoxins, protein drug conjugates exhibit improved therapeutic index over traditional chemotherapies with enhanced efficacy relative to standard monoclonal antibody therapies. However, current methods utilize nonspecific modes of conjugation of drugs to proteins, thereby leading to heterogeneous drug products with varied numbers of drugs conjugated across a number of possible sites. Technical challenges associated with drug conjugation to proteins using naturally occurring amino acids, are primarily due to heterogeneous degrees and location of drug loading as well as conjugate instability.
In order to reduce product heterogeneity, several groups have reported site-directed approaches that utilize substituted cysteines or enzymatic modification of engineered glutamine for conjugation. Site-specific ADCs have comparable potency to randomly conjugated ADCs while exhibiting superior therapeutic index and pharmacokinetics. However, limitations exist in thiol-based coupling stability due to plasma hydrolysis of the succinimde ring of the thiomaleimide conjugate, resulting in drug transfer to serum albumin. Furthermore, the partial reduction and reformation of disulfide bonds that facilitates conjugation to the engineered free cysteine, can lead to aberrant disulfide-mediated quaternary structure. An alternative to using introduced free cysteine residues is to use site-specific incorporation of non-natural amino acids with chemical side chains that are compatible with bio-orthogonal conjugation chemistry.
The essential componentry of any non-natural amino acid (nnAA) incorporation system consists of an aminoacyl tRNA synthetase (aaRS) that charges a specific tRNA with a nnAA. The aaRS-tRNA pair must be orthogonal with respect to the host cell or expression system in which they are employed. That is, the nnAA-specific synthetase must not recognize any host tRNAs or cognate amino acids, and the tRNA must not be aminoacylated by any host aaRS. Additionally, the orthogonal tRNA anticodon is often mutated to recognize a stop or nonsense codon. Repurposing of non-proteinogenic codons, such as the amber stop codon TAG, enables incorporation of a nnAA at any site in a protein through mutagenesis of the mRNA coding sequence to TAG. Amber suppression is the most widely used mode of co-translational, enzyme catalyzed nnAA incorporation. Non-natural amino acids with bio-orthogonal reactive chemical side chains can be used as a chemical “handle” to conjugate various payloads to discrete sites in a protein. This approach can generate additional functionality to proteins by direct conjugation of biologically active adduct, such as fluorescent or radioactive labels, photoactivatable markers, pharmacokinetic modifying PEGs, or chemotherapeutic agents. Unfortunately, the current methods for nnAA incorporation and conjugation of bio-orthogonal reactive chemical side chains into proteins are hindered by low overall product yield, nnAA incorporation inefficiency and low conjugation efficiency.
There is a need in the art for improved methods of site-specific incorporation of nnAAs and conjugation of biologically active adducts to proteins to form homogeneously conjugated protein drug conjugate therapeutics. The present invention satisfies these and other needs.